Device and method for mixing and bubble removal

ABSTRACT

A magnetic mixing device designed to mix fluid in a reaction chamber and remove air bubbles if present. The device comprises a holder with embedded magnets, which causes movement of a stir bar within the reaction chamber. The holder may be moved by an electric linear actuator configured to generate linear motion or an electric motor configured to generate a circular motion. When orientated so the stir bar moves vertically within the reaction chamber, the stir bar disrupts any air bubbles trapped within or below the fluid.

PRIORITY CLAIM

This application claims priority from U.S. Provisional PatentApplication No. 62/213,669 filed on Sep. 3, 2015, which is herebyincorporated by reference in its entirety in the present application.

BACKGROUND Technical Field

The present disclosure relates to the field of mixing devices and mixingmethods, and in particular, to a magnetic mixing device and method forusing a magnetic spin bar to circulate fluid and remove air bubbleswithin a reaction chamber.

Background

The mixing of solutions is routinely used in many industrial processesand is often essential in some chemical and biological reactions. Mixingis beneficial in any chemical or biological reaction where an equal andhomogeneous concentration is needed throughout the solution.

In biotechnology, mixing is generally performed by methods such aspipette mixing or vortexing. There are advantages and disadvantages ofeach method, for example, mixing may yield variability between samples.

Biotechnology companies use extensive amounts of culture media, buffers,and reagents. Such materials originally come in powdered form and mustbe rehydrated prior to use. Rehydrating the reaction components reducesreaction time and improves consistency between reactions.

In one area of biotechnology where rehydration of reaction components isdone for nucleic acid amplification, the reaction components may berehydrated by pipette mixing or vortexing, or thermal mixing before thereaction begins. Pipette mixing requires both specialized laboratoryskills and specialized laboratory equipment. Vortexing requiresspecialized laboratory equipment that is not typically battery operatedor portable. Both vortexing and pipette mixing are variable, dependingon the manner in which the operator performs the process. For example,there may be variability in the number of times the fluid is cycledthrough the micropipette or the duration of vortexing. Thermal mixingoccurs during the temperature cycling, however optimal amplification maybe delayed a few cycles until the solution is properly mixed. Thermalmixing is time-consuming, often taking approximately 10 minutes for thereaction solution to become thoroughly mixed. When using the invention,all parameters of the process are controlled, for example motor ramp uprate, motor revolutions per minute, motor duration at maximumrevolutions per minute, and motor ramp down rate.

Air bubble(s) may get trapped within or beneath the fluid.Traditionally, the air bubble is removed by methods such as acentrifugation or pipette mixing. Another common method of removing airbubbles is tapping the reaction plate or the tube. In a magnetic mixerwhere the stir bar rotates at the bottom of the reaction chamber, theair bubble may not be disrupted and may remain at the bottom of thereaction chamber. An air bubble can cause inconsistent results for anumber of reasons including reducing the effective volume of thereaction, preventing the reaction from achieving the appropriatereaction temperatures, interfering with a detector, and preventing thecomplete mixing of reaction components.

A device that quickly rehydrates dried reaction components, producesevenly distributed mixing throughout the reaction volume, and removesair bubbles in a reaction chamber is highly desirable. It is alsoadvantageous if this device is designed so any operator can use it, soit does not require a trained technician. Therefore, it is desirablethat the device be configured to alert a user if the device is notoperating properly.

SUMMARY

The present disclosure is directed to a magnetic mixer that can be usedfor mixing in any chemical or biological application. It is to beunderstood that the term “mix” in this disclosure refers to any movementthat creates a uniform solution, e.g. mix, stir, blend, agitate, etc.

It is to be understood that the term “holder” in this disclosure refersto any mechanical expedient, e.g. support, spindle, bracket, prop, etc.

Consistent with a disclosed embodiment, a device is disclosed thatquickly mixes solutions and removes air bubbles that may be present inthe reaction chamber. One application of the device and method of thepresent disclosure is the quick rehydration of dried reaction componentsby a magnetic mixer. In a nucleic acid amplification assay, for example,the dried down reaction components must be rehydrated to re-suspendreagents, reduce reaction time and improve consistency betweenreactions. Dried down reaction components may include, but are notlimited to, polymerase chain reaction (PCR) primers, PCR probes,nucleotides, taq polymerase, magnesium chloride, Bovine Serum Albumin,trehalose, and PCR buffer. Dried reaction components may also include,but not be limited to, NASBA, RPA, HDA, LAMP, RCA, ICAN, SMART, SDA, andLDR reaction components.

The geometry of a reaction chamber could cause air bubble(s) to betrapped within or beneath the fluid. The present disclosure describesdevices and methods that address both challenges of quick mixing andremoving trapped air bubbles.

Consistent with an exemplary embodiment of the present disclosure, adevice is disclosed that detects when a magnetic stir bar is movingwithin a reaction chamber. A control unit detects a change in current toa motor due to the presence of a magnetic stir bar and a feedback loopprovides data to a magnetic mixing device or a master instrument. A lackof change in current to a motor represents the absence of a magneticstir bar within a reaction chamber. The feedback loop to a masterinstrument can alert an untrained user to the absence of the magneticstir bar and any other potential problems with the magnetic mixingdevice.

Additionally, a device is disclosed that contains at least two magneticstir bars of a shape that promotes a grinding-type action to breakdown asample.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various embodiments and togetherwith the description, serve to explain the principles of the variousaspects of the embodiments. Other embodiments of this disclosure aredisclosed in the accompanying drawings, description, and claims. Thus,this summary is exemplary only, and is not to be considered restrictive.

BRIEF DESCRIPTION OF DRAWING(S)

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate the disclosed embodiments andtogether with the description, serve to explain the principles of thevarious aspects of the disclosed embodiments. In the drawings:

FIG. 1 illustrates a cross-sectional view of an exemplary embodiment ofan exemplary magnetic mixer of the present disclosure;

FIGS. 2A and 2B illustrate the complete mixing of the dried componentswithin the reaction chamber before and after mixing;

FIGS. 3A and 3B illustrate an air bubble trapped by the introduction ofa liquid, represented by hatched lines, to the reaction chamber and theremoval of the air bubble after mixing;

FIG. 4 illustrates the logic of a master instrument controlling themagnetic mixing device, in accordance with an exemplary embodiment ofthe present disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments consistentwith the present disclosure, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same reference numberswill be used throughout the drawings to refer to the same or like parts.

The present disclosure describes a magnetic mixing device. Exemplaryembodiments may include a rare earth magnet that attracts and/or repelsa paramagnetic stir bar.

The present disclosure further describes a device that removes trappedair bubbles beneath or within a fluid.

FIG. 1 illustrates an exemplary magnetic mixing device 10 comprising asource of motive power 20, a holder 30 containing an embedded magnet 40,a reaction chamber 50, stir bar(s) 60, housed in an outer housing 70.The stir bar(s) 60 is co-located within the reaction chamber 50.

In some embodiments, source of motive power 20 may be an electric motor,which in an illustrative embodiment is configured to generate a circularmotion. In alternative embodiments, source of motive power 20 may be anelectric linear actuator configured to generate linear motion. In otherembodiments, the movement of magnet 40 may be performed without a motoror a linear actuator, for example, by hand or other manual means. Thesource of motive power 20 can also be mixed-mode, relying on more thanone motor, actuator, manual motion, etc.

Another aspect of the present disclosure is holder 30 that contains oneor more embedded magnet 40. In an exemplary embodiment, holder 30 isembedded with one rare earth magnet 40. In alternative embodiments,holder 30 has more than one rare earth magnet 40 embedded in holder 30.In alternative embodiments, magnet 40 is another type of permanentmagnet. In alternative embodiments, magnet 40 is an electromagnet. Inalternative embodiments, holder 30 itself is a permanent magnet or anelectromagnet. In alternative embodiments, magnet 40 is moved relativeto reaction chamber 50 without being embedded in a holder.

Reaction chamber 50 may be configured for various functionalities. In anexemplary embodiment, reaction chamber 50 may be optimized to perform anucleic acid amplification reaction. In another exemplary embodiment,reaction chamber 50 may be contained within cartridge 90.

Another aspect of the present disclosure is one or more magnetic stirbar 60. The motion of stir bar 60 is driven by the movement of magnet40. Exemplary shapes of stir bar 60 may include disc, rod, cross, ring,and any other shape or construction capable of mixing. The size of stirbar 60 should be coupled to the size of reaction chamber 50 to allowstir bar 60 to move unrestricted.

In exemplary embodiments, stir bar 60 may be stainless steel, thoughstir bar 60 may be any magnetic or paramagnetic material. The magneticmaterial of stir bar 60 may be coated or uncoated. In exemplaryembodiments, stir bar 60 is coated with a material that does not reactwith or contaminate the reaction components. In an exemplary embodiment,stir bar 60 may be coated with parylene. In alternative embodiments, thecoating may be any number of coatings other than parylene. In suchembodiments, a preferred coating is one that has been FDA approved foruse in food, drug, and cosmetic applications.

Another aspect of the present disclosure is housing 70, which is used toalign source of motive power 20 and reaction chamber 50.

Holder 30 orientation relative to reaction chamber 50 may be in anymanner that allows for movement of stir bar 60 within reaction chamber50. In one embodiment, holder 30 is rotated parallel relative toreaction chamber 50. In another embodiment, holder 30 is rotatedperpendicularly relative to reaction chamber 50, along the longitudinalaxis of reaction chamber 50. In another embodiment, mixing device 10 isconfigured to allow reaction chamber 50 to sit at the center and holder30 moves magnet 40 around reaction chamber 50. In an exemplaryembodiment, holder 30 may be orientated to rotate alongside of reactionchamber 50 in a manner so that stir bar 60 moves vertically withinreaction chamber 50.

FIG. 2A illustrates the introduction of dried components 110 and fluid100 within a reaction chamber 50. FIG. 2B illustrates the resultinguniform solution 120 after mixing in a magnetic mixing device of thepresent disclosure.

An air bubble 80 may be trapped at the bottom of reaction chamber 50when fluid is introduced, as shown in FIG. 1 and FIG. 3A. In anorientation where stir bar 60 rotates at the bottom of reaction chamber50, trapped air bubble 80 may not be removed by motion of stir bar 60.In an orientation that moves stir bar 60 vertically within reactionchamber 50, air bubble 80 trapped at the bottom of a narrow reactionchamber 50 can be disrupted and/or air bubble 80 can be forced to thetop of reaction chamber 50.

FIG. 3A illustrates air bubble 80 trapped in a solution caused by theintroduction of a liquid, represented by hatched lines, to reactionchamber 50 within cartridge 90. FIG. 3B illustrates the removal of theair bubble after mixing using a magnetic mixing device of the presentdisclosure.

In an embodiment where the fluid has a viscosity high enough that airbubbles 80 may be trapped within the fluid, such as a gel or cream, anorientation that moves stir bar 60 vertically within reaction chamber 50may similarly disrupt air bubbles 80.

Further, an orientation that moves stir bar 60 vertically withinreaction chamber 50 may result in a turbulent flow, rather than apredictable vortex. The vertical motion advantageously creates an evenlydistributed mixing throughout the reaction volume.

It will be apparent to those skilled in the art that in embodimentswhere stir bar 60 is of a paramagnetic material, the distance betweenmagnet 40 and stir bar 60 must be within range to effectively attract orrepel stir bar 60 to cause the desired movement of stir bar 60 withinreaction chamber 50.

In an embodiment where there is more than one reaction chamber 50,corresponding stir bar(s) 60 must be in each reaction chamber 50. Theymust be positioned adjacent to magnet 40 within a range to effectivelyattract or repel stir bars 60 to cause the desired movement withinrespective reaction chambers 50.

In an alternative embodiment, magnet 40 may be stationary without theuse of a holder, and reaction chamber 50 may be moved relative to magnet40 by source of motive power 20 in the form of an electric motor orother manual means.

The present disclosure may contain more than one magnetic stir bar 60,within reaction chamber 50, of a shape to optimize a grinding-typeaction to break down a sample.

In an alternative embodiment, a microprocessor controls the ramp rateand speed of the source of motive power 20, for instance a motor, andtherefore stir bar 60. In an exemplary embodiment a master control unitcontrols magnetic mixing device 10 and selects a mixing protocol. Thecontrol unit may be part of a larger master instrument.

In such an embodiment, a control unit is configured to detect ifmagnetic stir bar 60 is within reaction chamber 50. A current necessaryto run a motor serving as the source of motive power 20 without thepresence of magnetic stir bar 60 is known. A control unit detects adifference in that current when magnetic stir bar 60 is present andmoving within reaction chamber 50. A lack of a change in current mayrepresent an absence of magnetic stir bar 60 within reaction chamber 50.A feedback loop provides input to magnetic mixing device 10 or a masterinstrument. An alert may be provided to a user regarding the absence ofmagnetic stir bar 60 and of any other potential problems with themagnetic mixing device.

In an alternative embodiment, a master device controls magnetic mixingdevice 10 as a slave device. In an exemplary embodiment, the masterdevice is a master instrument that controls magnetic mixing device 10.FIG. 4 illustrates an exemplary logic of a master instrument incontrolling a magnetic mixing device 10. By way of background, when anassay is developed, an optimal thermal protocol, mixing protocol, andresults interpretation methodology is prescribed. These pieces ofinformation can be advantageously encoded onto an information carrier,else contained in a database that can be referenced by indicia. Theinformation carrier can be in an illustrative embodiment a bar code,such as a 2-D barcode, which can contain, or provide the location ofsuch information, for example a thermal protocol ID, a mixing protocolID, a results interpretation ID, the manufacturing lot number of theassay, the catalog number of the assay, etc. At step 130, theillustrative master instrument reads a bar code. At step 140, the masterinstrument chooses a mixing protocol. This can be accomplished, forinstance, by reference to an online or offline database and/orfilesystem to retrieve the information referred to by the ID number. Atstep 150, the master instrument sends commands to magnetic mixing device10. At step 160, magnetic mixing device 10 sends a reply to the masterinstrument. In exemplary embodiments, the instrument may communicatewith the bar code module over USB, by wired or by wirelesscommunications. The command structure in an exemplary embodiment may bemodified as necessary to communicate with the bar code module employed.However any other communication protocol could be used, for instanceserial, RS232, RS485, SPI, I2C, WiFi direct, Bluetooth, etc. Imagingsystems such as cameras are also envisioned for capturing data labels,for example QR codes. RFID or other electromagnetic-based informationtags can also be used to encode the information described above in placeof an optical system.

Another embodiment of the present disclosure provides a kit foramplifying DNA from dried reaction components. The kit may comprise amagnetic mixing device as described above, dried down reactioncomponents, and a thermocycler for DNA amplification.

An exemplary method of mixing by magnetic mixing device 10 will now bedescribed. In describing the exemplary method, it will be assumed that auser is operating magnetic mixing device 10 shown in FIG. 1. However, itshould be understood that an automated, semi-automated, or manuallyoperated machine could also operate device 10 in a similar manner.

A user may fill reaction chamber 50 with the desired substances to bemixed, provided there is at least one substance that is a fluid. Atleast one magnetic stir bar 60 must be inserted into reaction chamber50, co-located with the substances to be mixed. Through movement ofmagnet 40 relative to a stationary reaction chamber 50 or movement ofreaction chamber 50 relative to a stationary magnet 40, magnetic stirbar 60 moves within reaction chamber 50 to create turbulent flow anduniform mixing throughout. Alternatively, a user may receive reactionchamber 50 containing at least one of the desired substances to bemixed, and/or magnetic stir bar 60. Reaction chamber 50 may be containedwithin cartridge 90. In an embodiment where magnetic stir bar 60 movesvertically throughout reaction chamber 50, any air bubbles 80 presentwithin or beneath the fluid are disrupted and/or moved to the top ofreaction chamber 50.

In an exemplary embodiment, a microprocessor may control the ramp rateand the speed of a motor serving as the source of motive power 20.

In an exemplary embodiment where magnetic mixing device 10 is used fornucleic acid amplification, a user fills reaction chamber 50 with driedreaction components 110 and a liquid. Through the use of magnetic mixingdevice 10, the dried reaction components 110 are rehydrated in theliquid. Alternatively, the user may receive reaction chamber 50 withdried reaction components 110 and magnetic stir bar 60 already withinreaction chamber 50. Reaction chamber 50 may be contained withincartridge 90.

In an exemplary embodiment, magnetic mixing device 10 may be used beforethe reaction chamber is placed in a master instrument.

In an alternative embodiment, magnetic mixing device 10 may be usedduring the run on a master instrument. At a specified time, the user mayremove the reaction chamber from the master instrument, place thereaction chamber in magnetic mixing device 10, run a mixing protocol,and place the reaction chamber back in the master instrument.

In an alternative embodiment, one or more magnetic mixing devices may beincorporated as part of a master instrument, rather than as an accessorythat communicates with a master instrument.

Table 1 presents data from a set of experiments where the magneticmixing device was used for nucleic acid amplification. The dataindicates the cycle threshold comparison between magnetic mixing,thermal mixing, and pipette mixing. The cycle threshold is the number ofcycles of amplification required to cross a threshold value. The datashows magnetic mixing is equal to or better than conventional thermalmixing or pipette mixing, with the added advantages of being faster andallowing an unskilled user to operate the magnetic mixing device.

TABLE 1 Comparison between Magnetic Mixing, Thermal Mixing, and PipetteMixing Magnetic Thermal Pipette Assay Mixing Mixing Mixing Type 1 29.430.1 30.4 Type 1 30.3 30.4 30.1 Type 2 33.8 36 not tested Type 2 34.236.1 not tested Type 1 31.4 33.7 not tested Type 1 30.8 33.8 not testedType 1 31.4 35 not tested Type 1 31.3 33 not tested Type 1 29.9 30.7 nottested Type 2 37.5 not tested 36.8 Type 2 37.4 not tested 37.2 Type 237.7 not tested 37.9 Type 2 37.3 not tested 37.9 Type 2 35.5 not tested38.4 Type 2 38.3 not tested 37.8 Type 2 37.2 not tested 38.2 Type 2 37.9not tested 38.5

The foregoing description has been presented for purposes ofillustration. It is not exhaustive and is not limited to the preciseforms or embodiments disclosed. Exemplary embodiments have beenpresented as being used for nucleic acid amplification, this disclosureis not limited to nucleic acid amplification and can be used for mixingin any chemical or biological application. Other embodiments will beapparent to those skilled in the art from consideration of thespecification and practice of the embodiments disclosed herein.

Moreover, while illustrative embodiments have been described herein, thescope of any and all embodiments include equivalent elements,modifications, omissions, combinations (e.g., of aspects across variousembodiments), adaptations and/or alterations as would be appreciated bythose skilled in the art based on the present disclosure. Thelimitations in the claims are to be interpreted broadly based on thelanguage employed in the claims and not limited to examples described inthe present specification or during the prosecution of the application.The examples are to be construed as non-exclusive. It is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the embodiments being indicated by thefollowing claims and their full scope of equivalents.

What is claimed is:
 1. A magnetic mixing device comprising at least onereaction chamber; at least one magnetic stir bar within each reactionchamber; at least one magnet magnetically coupleable to the stir bar andadjacent to the reaction chamber; a source of motive power positionedadjacent to the reaction chamber causing movement of the magnet andtherefore the magnetic stir bar.
 2. The magnetic mixing device of claim1, wherein source of motive power is an electric motor.
 3. The magneticmixing device of claim 1, wherein the source of motive power is anelectric linear actuator.
 4. The magnetic mixing device of claim 1,wherein said device is configured for the user to manually supply thesource of motive power.
 5. The magnetic mixing device of claim 1,wherein the at least one magnet is a permanent magnet.
 6. The magneticmixing device of claim 1, wherein the at least one magnet is anelectromagnet.
 7. The magnetic mixing device of claim 1, furthercomprising at least one magnet holder containing said at least onemagnet.
 8. The magnetic mixing device of claim 1, wherein said reactionchamber is contained within a cartridge.
 9. The magnetic mixing deviceof claim 1, wherein at least one magnetic stir bar is stainless steel.10. The magnetic mixing device of claim 1, wherein at least one magneticstir bar is a paramagnetic material.
 11. The magnetic mixing device ofclaim 1, wherein at least one magnetic stir bar is coated.
 12. Themagnetic mixing device of claim 11, wherein the magnetic stir bar is atleast partially coated with parylene.
 13. A method of mixing, comprisingapplying a force to move a holder with embedded magnets, causingmovement of a magnetic spin bar within a reaction chamber; wherein themovement of the stir bar creates a uniformly mixed solution and disruptsair bubbles.
 14. The method of claim 13, wherein the movement caused bythe applying force is a linear movement of the magnetic spin bar. 15.The method of claim 13, wherein the movement caused is horizontalrelative to a long axis of the reaction chamber.
 16. The method of claim13, wherein the movement caused is vertical relative to a long axis ofthe reaction chamber.
 17. The method of claim 13, further comprising thesteps of: filling the reaction chamber with reaction components,including at least one fluid; and inserting at least one magnetic stirbar in the reaction chamber.
 18. The method of claim 13, furthercomprising the step of: receiving the reaction chamber with the reactioncomponents and at least one magnetic stir bar inside.
 19. The method ofclaim 13, wherein said step of applying a force further comprisesapplying force from an electric motor.
 20. The method of claim 13,wherein said step of applying a force further comprises applying forcefrom an electric linear actuator.
 21. The method of claim 13, whereinsaid step of applying a force further comprises manually applying force.22. A kit for amplifying DNA from dried reaction components, the kitcomprising (a) a magnetic mixing device, (b) dried down reactioncomponents, and (c) a thermocycler for DNA amplification.